The present invention relates a novel glutaminase and a gene encoding the same. The glutaminase of the present invention can be utilized as an enzyme for food processing to convert glutamine into glutamic acid exhibiting stronger xe2x80x9cumamixe2x80x9d taste (umami).
For the production of soy sauce, miso, and other natural seasonings containing protein hydrolysate products, koji mould (filamentous fungus belonging to the genus Aspergillus) has been utilized. For example, soy sauce is produced through two process steps of koji-making and fermentation. In the koji-making step, the starting material is principally degraded by enzymes produced by koji mould. In such a process, it is important to increase the amount of glutamic acid among various tasteful materials in order to obtain stronger umami of soy sauce.
Glutamic acid is produced through two kinds of pathways. The first is the liberation of glutamic acid from protein caused by protease and peptidase. The second is generation of glutamic acid through hydrolysis of glutamine catalyzed by glutaminase (glutamine amidohydrolase).
In the production of soy sauce, liberation ratio of glutamic acid relative to its content in the starting material is not so high, and this is considered to be due to insufficient glutaminase activity of koji mould. Therefore, breeding of strains exhibiting high activities of protease and glutaminase through cell fusion of high protease activity strain and high glutaminase activity strain in solid koji has also been attempted (Ushijima, S. et al., Agric. Biol. Chem., 51 (4), 1051 (1987), Japanese Patent Publication (KOKOKU) No. Hei 3-73271/1992).
As for glutaminase, those derived from various bacteria and animals have been well investigated (Wakayama, M. et al., J. Ferment. Bioeng., 82, No.6, 592-597 (1996), Chung-Bok, Mi, et al., Biochem. J., 324, 193-200 (1997), Duran, S. et al., Biochem. Genet., 34, 453-465 (1996)). On the other hand, investigation about glutaminase of koji mould had been retarded, but extracellular glutaminase and intracellular glutaminase have been purified from one strain of Aspergillus oryzae, and they have been characterized (Yano, T. et al., J. Ferment. Technol., Vol. 66, No. 2, 137-143 (1988)). These glutaminases have a molecular weight of about 113,000, and substantially similar properties.
Further, there have been determined an amino acid sequence of N-terminal region of glutaminase derived from Aspergillus oryzae HG strain (Fukuoka Industrial Technology Center, Institute of Biology and Food, Research Summary of 1996 (199)), and amino acid sequence within N-terminal region of glutaminase derived from Aspergillus oryzae (Food Research Institute, Aichi Prefectural Government, Japan, Annual Report of 1995 (Research Report) pp.3-4, (1996)) for purified glutaminases.
Meanwhile, because koji mould is excellent in the ability for secreting extracellular proteins, it has been attracted attention as a host for the production of recombinant proteins, and practically used for some enzymes.
As described above, koji mould has already afforded results as a material for genetic recombination technology, and its glutaminase has also been investigated to some extent. However, it cannot be considered to be fully investigated, and its further investigation has been desired. In addition, any genes encoding glutaminase of koji mould have not been isolated.
The present invention has been accomplished in view of the aforementioned state of the art, and its object is to provide a gene encoding glutaminase derived from koji mould.
The present inventors successfully purified glutaminase from Aspergillus oryzae, determined its partial amino acid sequence, and isolated DNA coding for the glutaminase based on the obtained information, and thus the present invention has been completed. Further, they also succeeded in isolating DNA encoding glutaminase of Aspergillus nidulans. 
That is, the present invention provides the followings:
(1) a protein defined in any of the following (A) to (D):
(A) a protein having an amino acid sequence represented by the amino acid numbers 1-670 of SEQ ID NO: 2 in Sequence Listing;
(B) a protein having an amino acid sequence represented by the amino acid numbers 1-669 of SEQ ID NO: 22 in Sequence Listing;
(C) a protein having an amino acid sequence represented by the amino acid numbers 1-670 of SEQ ID NO: 2 in Sequence Listing with substitution, deletion, insertion, addition or inversion of one or a plurality of amino acids, and having activity for catalyzing hydrolysis of glutamine to glutamic acid and ammonia;
(D) a protein having an amino acid sequence represented by the amino acid numbers 1-669 of SEQ ID NO: 22 in Sequence Listing with substitution, deletion, insertion, addition or inversion of one or a plurality of amino acids, and having activity for catalyzing hydrolysis of glutamine to glutamic acid and ammonia;
(2) a DNA which encodes a protein defined in any of the following (A) to (D):
(A) a protein having an amino acid sequence represented by the amino acid numbers 1-670 of SEQ ID NO: 2 in Sequence Listing;
(B) a protein having an amino acid sequence represented by the amino acid numbers 1-669 of SEQ ID NO: 22 in Sequence Listing;
(C) a protein having an amino acid sequence represented by the amino acid numbers 1-670 of SEQ ID NO: 2 in Sequence Listing with substitution, deletion, insertion, addition or inversion of one or a plurality of amino acids, and having activity for catalyzing hydrolysis of glutamine to glutamic acid and ammonia;
(D) a protein having an amino acid sequence represented by the amino acid numbers 1-669 of SEQ ID NO: 22 in Sequence Listing with substitution, deletion, insertion, addition or inversion of one or a plurality of amino acids, and having activity for catalyzing hydrolysis of glutamine to glutamic acid and ammonia;
(3) the DNA of (2) which is a DNA defined in any of the following (a) to (d):
(a) a DNA which contains nucleotide sequences represented by the nucleotide numbers 1174-1370, 1446-1741, 1800-2242, 2297-2880, 2932-3134, 3181-3324, 3380-3515, 3562-3628 of the nucleotide sequence or SEQ ID NO: 1 in Sequence Listing in this order;
(b) a DNA which contains nucleotide sequences represented by the nucleotide numbers 1807-2000, 2061-2353, 2412-2854, 2915-3498, 3554-3756, 3806-3949, 3996-4131, 4180-4246 of the nucleotide sequence of SEQ ID NO: 21 in Sequence Listing in this order;
(c) a DNA which hybridizes with the DNA of (a) under a stringent condition, and encodes a protein having activity for catalyzing hydrolysis of glutamine to glutamic acid and ammonia;
(d) a DNA which hybridizes with the DNA of (b) under a stringent condition, and encodes a protein having activity for catalyzing hydrolysis of glutamine to glutamic acid and ammonia;
(4) the DNA of (2) which has a nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 17;
(5) the DNA of (3) which has a nucleotide sequence shown in SEQ ID NO: 21 or SEQ ID NO: 25;
(6) a recombinant vector comprising the DNA of (2) inserted in a vector;
(7) a transformant of microorganism introduced with the DNA of (2) in such a manner that the DNA can be expressed to produce glutaminase;
(8) the transformant of (7) which is derived From a filamentous fungus or bacterium belonging to the genus Escherichia; and
(9) a method for producing glutaminase which comprises cultivating the transformant of (7) in a culture medium to produce glutaminase in the culture.
The term xe2x80x9cglutaminase activityxe2x80x9d used in this specification means activity for catalyzing hydrolysis of L-glutamine to L-glutamic acid and ammonia, and the activity may include activity for catalyzing hydrolysis of D-glutamine to D-glutamic acid and ammonia. The activity may also include activities for catalyzing hydrolysis of L-glutamine to L-glutamic acid and ammonia, and D-glutamine into D-glutamic acid and ammonia, and activity for catalyzing transfer reaction or hydrolysis reaction of glutamyl group of L-xcex3-glutamyl compounds. In the present specification two embodiments are disclosed as the glutaminase of the present invention. One or both of the embodiments, or equivalents thereof may occasionally be referred to as glutaminase of the present invention. Also, the DNA which encodes the glutaminase of the present invention may occasionally be referred to as glutaminase gene.
The glutaminase of the present invention is distinguished from known glutaminases derived from koji mould based on enzymological properties, and therefore it is considered a novel glutaminase.
The present invention will be explained in detail hereinafter.
 less than 1 greater than  Glutaminase of the Present Invention
The glutaminase of the present invention can be obtained from culture of Aspergillus oryzae RIB40 (ATCC 42149) by purifying it, for example, as follows.
Aspergillus oryzae RIB40 (ATCC 42149) is cultured with wheat bran, and the obtained bran koji is immersed in a buffer solution to prepare a crude enzyme extract. This crude enzyme extract is subjected to freeze and thawing, and insoluble fractions are removed to obtain a supernatant. This supernatant is subjected to ammonium sulfate fractionation to obtain a fraction not precipitated with 55% saturated ammonium sulfate but precipitated with 85% saturated ammonium sulfate. The ammonium sulfate is removed from this fraction, and resultant can further be fractionated by anion exchange chromatography, hydrophobic chromatography, and gel filtration chromatography to provide purified glutaminase. As resins for the chromatographies, there are exemplified DEAE-TOYOPEARL (Tosoh) for the anion exchange chromatography, Phenyl Sepharose (Pharmacia) for the hydrophobic chromatography, and Superdex (Pharmacia) for the gel filtration chromatography. These purification procedures may be repeatedly performed.
In each step for purification or glutaminase, the desired fraction is selected based on the glutaminase activity. The glutaminase activity can be determined by a modified version of the method of Hartaran (Hartman, S. C., J. Biol. Chem., 243, 853-863 (1968), the hydroxamate method).
Enzymological properties of the glutaminase obtained from bran koji of Aspergillus oryzae RIB40 (ATCC 42149) as described above are shown in Table 1 together with enzymological properties of known glutaminases, one derived from of Aspergillus oryzae (Yano, T. et al., J. Ferment. Technol., Vol. 66, No. 2, 137-143 (1988)), and one derived from Bacillus subtilis (Shimazu, H. et al., J. Brew. Soc. Japan, 86, No. 6, 441-446 (1991)).
Based on the marked differences in enzymological properties shown above, in particular in the substrate specificity, the glutaminase of the present invention is concluded to be novel, and different from the known glutaminase derived from Aspergillus oryzae. 
While the glutaminase of the present invention can be obtained by purifying it from culture of Aspergillus oryzae as described above, it can also be produced by expression of glutaminase gene of Aspergillus oryzae described below in a suitable host as will be described hereinafter.
As will be described hereinafter, the glutaminase derived from Aspergillus oryzae is expected to have the amino acid sequence represented by the amino acid numbers 1-670 in SEQ ID NO: 2 based on the nucleotide sequence of glutaminase gene. The molecular weight calculated from this amino acid sequence is about 76,000, and from its comparison with the value of molecular weight measured by MALDI-TOFMS, the glutaminase of the present invention is expected to be a glycoprotein.
The glutaminase of another embodiment of the present invention is derived from Aspergillus nidulans. The glutaminase derived from Aspergillus nidulans may be produced by purifying from a culture of Aspergillus nidulans in the same manner as described above, or expressing the glutaminase gene of Aspergillus nidulans in an appropriate host. The glutaminase derived from Aspergillus nidulans has deduced amino acid sequence represented by the amino acid numbers 1-669 of SEQ ID NO: 22 from the nucleotide sequence of the glutaminase gene.
As for the glutaminase of the present invention, so long as it has activity for catalyzing hydrolysis of glutamine to glutamic acid and ammonia, the aforementioned amino acid sequence may have substitution, deletion, insertion, addition or inversion of one or a plurality of amino acids.
The present invention also provides, as an embodiment of the glutaminase of the present invention, glutaminase of Aspergillus nidulans having the amino acid sequence shown in SEQ ID NO: 22. This glutaminase can be produced by purifying it from culture of Aspergillus nidulans in a manner similar to that described above, or by expression of glutaminase gene of Aspergillus nidulans in a suitable host.
 less than 2 greater than  DNA of the Present Invention
The DNA of the present invention can be obtained from genomic DNA of Aspergillus oryzae RIB40 (ATCC 42149), for example, as follows.
A partial amino acid sequence of the purified glutaminase is determined, and oligonucleotide primers for PCR (polymerase chain reaction) are synthesized based on the obtained information of the amino acid sequence to perform PCR using genomic DNA prepared from fungal cells of Aspergillus oryzae RIB40 (ATCC42149) as template. Partial sequences determined in the working examples of the present invention to be described hereinafter are shown in SEQ ID NOS: 3-10. Among these sequences, SEQ ID NO: 3 is an N-terminal amino acid sequence of the glutaminase protein, and the other sequences are internal amino acid sequences of the glutaminase. The amino acid sequences shown in SEQ ID NOS: 5 and 8 were not present in the amino acid sequence of glutaminase expected from the glutaminase gene. The third Ala and the ninth Thr in the amino acid sequence shown in SEQ ID NO: 7 were replaced by Thr and Ser respectively in the amino acid sequence of glutaminase expected from the glutaminase gene, and it was considered that they were reading errors in peptide sequencer.
The genomic DNA can be obtained by the method of Gomi (Gomi, K. et al., J. Gen. Appl. Microbiol., 35, 225 (1989)).
By using oligonucleotides having nucleotide sequences shown in SEQ ID NO: 11 and SEQ ID NO: 12 of Sequence Listing as the primers, a DNA fragment of about 230 bp can be obtained by the aforementioned PCR.
Then, plaque hybridization is performed for a genomic DNA library of Aspergillus oryzae RIB40 (ATCC 42149) utilizing xcex phage as a vector by using the DNA. fragment amplified by PCR as a DNA probe to obtain positive clones.
Within the cloned fragment obtained as described above, nucleotide sequence of a portion having a length of about 4 kb within a region having about 4.8 kb (XHoI fragment) is determined, and the result is shown in SEQ ID NO: 1 of Sequence Listing. In SEQ ID NO: 1, the amino acid sequence encoded by nucleotides of the nucleotide numbers 1234-1284 corresponds to the amino acid sequence of the amino acid numbers 1-17 in the N-terminal amino acid sequence of the glutaminase protein shown in SEQ ID NO: 3. The amino acid sequences shown in SEQ ID NOS: 4, 6, 7, 9 and 10 respectively correspond to the amino acid sequences encoded by nucleotides of the nucleotide numbers 2618-2647, 2762-2803, 2804-2848, 2957-2986, and 2576-2605 of the nucleotide sequence shown in SEQ ID NO: 1.
From the above, it is clear that DNA having the nucleotide sequence shown in SEQ ID NO: 1 is a glutaminase gene.
From the comparison of the nucleotide sequence shown in SEQ ID NO: 1 and the nucleotide sequence of glutaminase cDNA to be described hereinafter, it was found that the nucleotide sequence of SEQ ID NO: 1 contained 8 exons (nucleotide numbers 1174 or 1135-1370, 1446-1741, 1800-2242, 2297-2880, 2932-3134, 3181-3324, 3380-3515, and 3562-3628), and these exons encoded an amino acid sequence comprised of 690 residues. This amino acid sequence is shown in SEQ ID NOS: 1 and 2. From the comparison of this amino acid sequence and the amino acid sequence of the N-terminal of the glutaminase protein shown in SEQ ID NO: 3, it is estimated that the sequence of the amino acid numbers xe2x88x9220 to xe2x88x921 is a signal peptide, and the sequence of the amino acid numbers 1-670 is the mature protein in SEQ ID NO: 2. While the initiation codon is estimated to be ATG of the nucleotide numbers 1174-1176, the possibility that it consists of ATG at the nucleotide numbers 1135-1138 cannot be denied.
From the above, it is strongly suggested that DNA having the nucleotide sequence shown in SEQ ID NO: 1 contains a promoter and a region encoding glutaminase (including signal peptide).
The DNA of the present invention may be DNA of the nucleotide sequence shown in SEQ ID NO: 1 of which introns are removed, i.e., DNA comprising nucleotide sequences of nucleotide numbers 1174-1370, 1446-1741, 1800-2242, 2297-2880, 2932-3134, 3181-3324, 3380-3515 and 3562-3628 in this order, so long as it encodes the glutaminase of the present invention. Such DNA can be obtained, for example, as cDNA of the aforementioned glutaminase gene.
Glutaminase cDNA can be obtained, for example, from a cDNA library prepared from poly(A) RNA of Aspergillus oryzae by hybridization which utilizes DNA having the nucleotide sequence of SEQ ID NO: 1 or a part thereof (e.g., the aforementioned probe of about 230 bp).
Glutaminase cDNA can also be obtained by PCR utilizing oligonucleotides having the nucleotide sequences of SEQ ID NOS: 13 and 14 as primers, and by 3xe2x80x2-RACE utilizing oligonucleotides having the nucleotide sequences of SEQ ID NOS: 15 and 16 as primers. An exemplary nucleotide sequence of cDNA obtained from a highly glutaminase productive strain of Aspergillus oryzae is shown in SEQ ID NO: 17 of Sequence Listing. The amino acid sequence deduced from this nucleotide sequence is shown in SEQ ID NOS: 17 and 18. When the nucleotide sequence of this cDNA was compared with the sequence of the coding region in the genomic gene obtained in Example 2, they were identical except that xe2x80x9cCxe2x80x9d at the nucleotide number 54 of the cDNA (SEQ ID NO: 17) was xe2x80x9cGxe2x80x9d (nucleotide number 1227) in the genomic gene (SEQ ID NO: 1). This difference between the nucleotide sequences of the cDNA and the genomic gene is estimated to be due to difference of gene sequence between the strains.
The DNA of the present invention may be any one encoding glutaminase, and it includes, in addition to DNA having the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 17, those DNA of which unnecessary portions in 5xe2x80x2 region have been removed. Depending on purpose of the use, it may be one encoding only the mature protein. DNA of which one or more codons encoding amino acids in the coding region are replaced with equivalent codons encoding the same amino acids is included in the DNA of the present invention. Further, the DNA of the present invention may be one encoding glutaminase having substitution, deletion, insertion, addition or inversion of one or a plurality of amino acids at one or a plurality of sites, so long as the activity of glutaminase is not degraded. The number of the amino acid meant by the expression xe2x80x9ca plurality ofxe2x80x9d may vary depending on the location or kinds of amino acid residues in the three-dimensional structure of the glutaminase protein, but it may be usually 2-300, preferably 2-170, more preferably 2-50, most preferably 2-10.
As will be described hereinafter, the amino acid sequence of glutaminase of Aspergillus oryzae shown in SEQ ID NO: 2 and the amino acid sequence of glutaminase of Aspergillus nidulans shown in SEQ ID NO: 22 have about 73% of homology, and about 170 amino acid residues are different between them as for the mature protein portion.
DNA encoding a protein substantially the same as glutaminase such as those mentioned above can be obtained by modifying the nucleotide sequence of glutaminase gene, for example, by the site-specific mutagenesis so that amino acids should be substituted, deleted, inserted or added at a particular site. Such modified DNA as mentioned above may also be obtained by a conventionally known mutagenesis treatment. As such a mutagenesis treatment, there can be mentioned a method comprising treating DNA encoding glutaminase with hydroxylamine or the like in vitro, and a method comprising irradiating a bacterium belonging to the genus Escherichia with ultraviolet light, or treating it with a mutagenic agent conventionally utilized for mutagenesis such as N-methyl-Nxe2x80x2-nitro-N-nitrosoguanidine (NTG), and nitrous acid.
The substitution, deletion, insertion, addition and inversion mentioned above include those due to difference among strains, and naturally occurring mutations.
DNA encoding a protein substantially the same as glutaminase can be selected by expressing DNA having mutations as described above in a suitable cell, and examining the expression product for glutaminase activity. DNA encoding a protein substantially the same as glutaminase can also be obtained by isolating DNA which hybridizes with DNA having any one of nucleotide sequences of nucleotide numbers 1174-1370, 1446-1741, 1800-2242, 2297-2880, 2932-3134, 3181-3324, 3380-3515, and 3562-3628 in the nucleotide sequence of SEQ ID NO: 1 in Sequence Listing, or DNA having the nucleotide sequence of the nucleotide numbers 1-2070 in the nucleotide sequence of SEQ ID NO: 17 under a stringent condition, and encodes a protein having the glutaminase activity. The term xe2x80x9cstringent conditionxe2x80x9d herein used means a condition where so-called specific hybrids may be formed, but non-specific hybrids are not formed. While it is difficult to definitely define this condition numerically, examples of such condition include, for example, a condition where DNAs having high homology, e.g., homology of 65% or more may hybridize with each other, but DNAs having homology lower than that may not hybridize with each other, and a condition where hybridization is performed at a salt concentration corresponding to that of washing step of usual Southern hybridization, i.e., 1xc3x97SSC, 0.1% SDS, preferably 0.1xc3x97SSC, 0.1% SDS. Genes which hybridize under such a condition may also include those having a stop codon generated to interrupt the coding sequence, those having lost their activity due to mutation at the active center and the like, but they can easily be removed by ligating the genes to a commercially available active expression vector, and determining glutaminase activity by the method described hereinafter.
The DNA of the present invention can also be obtained from chromosome DNA or cDNA of microorganism of another species belonging to the genus Aspergillus, for example, Aspergillus nidulans. Specifically, it can be obtained from a chromosome DNA library of Aspergillus nidulans, for example, Aspergillus nidulans A26 strain by hybridization. A probe for the hybridization can be prepared by synthesizing oligonucleotide primers For PCR based on the aforementioned nucleotide sequence of the glutaminase gene of Aspergillus oryzae, and performing PCR using genome DNA prepared from cells of Aspergillus nidulans, e.g., Aspergillus nidulans A26 strain as template. As the primers for PCR, oligonucleotides having nucleotide sequences of SEQ ID NOS: 19 and 20 can be mentioned.
The nucleotide sequence and the amino acid sequence of the glutaminase gene of Aspergillus nidulans A26 obtained in the working examples to be described hereinafter in the manner described above are shown in SEQ ID NO: 21. The amino acid sequence is also shown in SEQ ID NO: 22. The homology between the glutaminase gene of Aspergillus nidulans and the glutaminase gene of Aspergillus oryzae was about 58% for the whole gene, about 68% for the coding region, and about 73% for the encoded amino acid sequence.
Glutaminase cDNA can also be obtained from a cDNA library prepared from poly(A) RNA of Aspergillus nidulans by, for example, PCR using oligonucleotides having nucleotide sequences SEQ ID NOS: 23 and 24. An exemplary nucleotide sequence of cDNA obtained from Aspergillus nidulans A26 is shown in SEQ ID NO: 25 of Sequence Listing. The amino acid sequence deduced From this nucleotide sequence is shown in SEQ ID NOS: 25 and 26.
The DNA of the present invention includes a DNA which encodes a protein having an amino acid sequence represented by the amino acid numbers 1-669 of SEQ ID NO: 22 in Sequence Listing with substitution, deletion, insertion, addition or inversion of one or a plurality of amino acids, and having activity for catalyzing hydrolysis of glutamine to glutamic acid and ammonia. The DNA of the present invention also includes a DNA which encodes a DNA which contains nucleotide sequences represented by the nucleotide numbers 1807-2000, 2061-2353 2353, 2412-2854, 2915-3498, 3554-3756, 3806-3949, 3996-4131, 4180-4246 of the nucleotide sequence of SEQ ID NO: 21 in Sequence Listing in this order, and a DNA which hybridizes with the aforementioned DNA under a stringent condition, and encodes a protein having activity for catalyzing hydrolysis of glutamine to glutamic acid and ammonia.
The DNA of the present invention was obtained as described above in the wording examples to be described hereinafter. However, since its nucleotide sequence has been elucidated, it can easily be cloned by PCR, hybridization or the like from genomic DNA of Aspergillus oryzae RIB40 (ATCC 42149), Aspergillus nidulans A26, or other strains of Aspergillus oryzae and Aspergillus nidulans. 
 less than 3 greater than  Use of the DNA of the Present Invention
The DNA of the present invention can be utilized for breeding of filamentous fungi such as koji mould or production of glutaminase. For example, glutaminase activity can be enhanced by intracellularly introducing the DNA of the present invention, preferably as its multiple copies, into filamentous fungus. Glutaminase can be produced by expressing the DNA of the present invention in a suitable host. A filamentous fungus such as koji mould and glutaminase obtained as described above can be utilized for the production of soy sauce, miso, and other seasonings containing protein hydrolysate products.
As the filamentous fungus to be introduced with the DNA of the present invention, there can be mentioned filamentous fungi belonging to the genus Aspergillus such as Aspergillus oryzae, Aspergillus niger and Aspergillus nidulans, those belonging to the genus Neurospora such as Neurospora crassa, those belonging to the genus Rhizomucor such as Rhizomucor miehei, and the like.
The vector for introducing the DNA of the present invention into filamentous fungi such as those mentioned above is not particularly limited, and those usually used for the breeding of filamentous fungi and the like can be used. As those used for Aspergillus oryzae, there can be mentioned, for example, pUNG (Lee, B. R. et al., Appl. Microbiol. Biotechnol., 44, 425-431 (1995)), pMARG (Tsuchiya, K. et al., Appl. Microbiol. Biotechnol., 40, 327-332 (1993)), pUSC (Gomi, K. et al., Agric. Biol. Chem. 51, 2549-2555 (1987)) and the like. pUNG contains a marker complementing niaD31  (nitrate assimilation ability defficiency) of Aspergillus oryzae niaD300 (Minetoki, T. et al., Curr. Genet. 30, 432-438 (1996)), pMARG contains a marker complementing argB31 (arginine auxotroph) of Aspergillus oryzae M2-3 (Gomi, K. et al., Agric. Biol. Chem., 51(9), 2549-2555 (1987)), and pUSC contains a marker complementing sC31  (ATP sulfurylase defficiency) of Aspergillus oryzae NS4 (Yamada, O. et al., Biosci. Biotech. Biochem., 61(8), 1367-1369 (1997))
Among these vectors, pUNG and pMARG contain a promoter of glucoamylase gene (glaA) and xcex1-amylase gene (terminator of amyB), and the DNA of the present invention (region of the nucleotide numbers 1136-4777 or 1177-4777 in SEQ ID NO: 1) can be expressed in them under the control of the promoter to produce glutaminase by inserting the DNA into them in the downstream of the promoter in such a manner that the frames should be conformed. When pUSC is used, because pUSC does not contain a promoter, expression of the gene of the present invention can be obtained by introducing a plasmid such as pUC19 inserted with the DNA of the present invention and pUSC into a host filamentous fungus through co-transformation of them. Since the nucleotide sequences of SEQ ID NO: 1 is likely to contain a promoter as described hereinbefore, it is considered that glutaminase can be expressed even if the DNA of the present invention is inserted into the aforementioned vector together with a promoter.
Those vectors, promoters and markers described in the literature mentioned below can also be used depending on the host filamentous fungus. In Table 2, promoters are indicated by the enzyme names encoded by corresponding genes.
Transformation of filamentous fungi can be performed by the methods mentioned in the aforementioned literature as well as other known methods. Specifically, Aspergillus oryzae for example, can be transformed as follows.
Fungal cells (conidiospores) are inoculated in DPY culture medium (2% glucose, 1% peptone, 0.5% yeast extract, pH 5.0), and cultured at 30xc2x0 C. for around 24 hours with vigorous shaking. The culture medium is filtered through Myracloth (CALBIO CHEM), sterilized gauze or the like to collect the fungal cells, the cells are washed with sterilized water, and moisture is sufficiently removed from the cells. The cells are transferred into a test tube, added with an enzyme solution (1.0% Yatalase (Takara Shuzo), or 0.5% Novozyme (Novo Nordisk) and 0.5% cellulase (e.g., Cellulase Onozuka, Yakult), 0.6 M (NH4)2SO4, 50 mM malic acid, pH 5.5), and gently shaken at 30xc2x0 C. for around 3 hours. The degree of protoplastization is observed with a microscope, and they are stored on ice if they show good protoplastization.
The aforementioned enzymatic reaction mixture is filtered through Myracloth to remove the fungal cell residue, and the filtrate containing protoplasts is added with an equal volume of Buffer A (1.2 M sorbitol, 50 mM CaCl2, 35 mM NaCl, 10 mM Tris-HCl, pH 7.5), and placed on ice. The mixture is centrifuged at 0xc2x0 C. and 2,500 rpm for 8 minutes, and gently stopped, and the pellet is washed with Buffer A, and suspended in an optimum volume of Buffer A.
A DNA solution of not more than 20 xcexcl (5-10 xcexcg) is added to 100-200 xcexcl of the protoplast suspension, and placed on ice for 20-30 minutes. To the mixture, 250 xcexcl of Buffer B (polyethylene glycol 6000, 50 mM CaCl2, 10 mM Tris-HCl, pH 7.5) is added and gently mixed, again 250 xcexcl of Buffer B is added and gently mixed, further 850 xcexcl of Buffer B is added and gently mixed, and then the mixture is left stand at room temperature for 20 minutes. Then, 10 ml of Buffer A is added to the mixture, and the test tube is inverted and subjected to centrifugation at 0xc2x0 C. and 2,000 rpm for 8 minutes. Subsequently, the pellet is suspended in 500 xcexcl of Buffer A.
A suitable amount of the above suspension is added to 5 ml top agar, which has been divided into fractions and warmed beforehand, overlaid on an under layer culture medium (selection medium containing 1.2 M sorbitol, which is prepared depending on the kind of marker), and cultured at 30xc2x0 C. Grown fungal cells are transferred on the selection medium, and confirmed to be transformants. Recombinant DNA is prepared from the fungal cells. It is preferable to confirm that the DNA of the present invention is introduced into the recombinant DNA by restriction enzyme analysis, Southern analysis or the like.
When the transformants obtained as described above are cultured under a condition suitable for the promoter used, the glutaminase gene is expressed, and thus glutaminase is produced.
By allowing a culture of transformants that are introduced with the gene of the present invention and have enhanced glutaminase activity to react with protein, protein hydrolysis products having higher sodium glutamate content and stronger umami can be afforded. Examples of the protein to be reacted with the culture include, for example, those of soybean, wheat, wheat gluten and the like, and it may be those of defatted soybean, or any one of various proteins subjected to food processing such as swelling and solubilization, or proteins isolated from these various kinds of materials.
As for the condition of the reaction of the culture of transformants with the protein, for example, a starting material having a concentration of 0.2-50% may be mixed with a culture of transformants in the presence of a proteolytic enzyme, and allowed to react at 5-60xc2x0 C. for 4 hours to 10 days.
After the completion of the reaction, insoluble unreacted proteins, fungal cells and the like can be removed by using conventional separation methods such as centrifugal separation or filtration. If required, the reaction mixture may be concentrated by vacuum concentration, reverse osmosis or the like, and the concentrate can be made into powder or granules by a drying process such as lyophilization, drying under reduced pressure, and spray drying. Thus, protein hydrolysates having high sodium glutamate content, and exhibiting stronger umami can be obtained without externally adding sodium glutamate.